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Abstract

The GloMax®-Multi+ Detection System with Instinct Software is a customizable multimode microplate reader that can be used for detecting fluorescence intensity, luminescence, visible absorbance and UV-visible absorbance. It combines the superior performance expected from single-mode instruments with the functionality of multiple modes. In this article, we demonstrate the versatility of the GloMax®-Multi+ Detection System by highlighting its capabilities.

Introduction

The GloMax®-Multi+ Detection System with Instinct Software is designed for the typical life science laboratory—it is easy to use, affordable and can be customized to a laboratory’s needs. The GloMax®-Multi+ Detection System with Instinct Software (Cat.# E8032, E9032) is a multimode microplate reader that accepts 6-well to 384-well plates and can be used for fluorescence, luminescence, visible absorbance and UV-visible absorbance applications.

Unlike many multimode instruments, the GloMax®-Multi+ Detection System performance is equivalent to that of single-mode instruments. It has a linear dynamic range >8 logs for luminescent assays and up to 6 logs for fluorescent assays, with optical channels dedicated to each individual technology. The system includes four snap-in fluorescent optical kits for wavelengths in the UV, blue, green and red ranges, as well as an optional optical kit for AFC measurement. It also includes six filters for UV-visible absorbance at 260, 280, 450, 560, 600, 750nm. The instrument contains a PC (personal computer) with a touch screen, so that an external computer is not required for operation. Data can be transferred using a USB flash drive. In addition, GloMax®-Multi+ Detection System instruments are supplied with Instinct software installed, allowing the user to analyze data using the touch screen.

The GloMax®-Multi+ Detection System is a modular instrument: Luminescence, fluorescence and absorbance modules can be purchased with the base unit or added after purchasing the instrument. The system uses customizable fluorescent optical kits or custom visible or UV-visible filters. The GloMax®-Multi+ Detection System also includes a factory-installed shaker that allows either linear or orbital shaking, while an optional heater may be purchased for precise temperature control from 2°C above ambient temperature to 45°C. Finally, the GloMax®-Multi+ Detection System with Instinct Software contains preloaded protocols and has the ability to create new protocols with multiple detection modes using the protocol composer. The following examples demonstrate several of the many options and functions of the GloMax®-Multi+ Detection System with Instinct Software.

"The GloMax®-Multi+ Detection System is a modular instrument: Luminescence, fluorescence and absorbance modules can be purchased with the base unit or added after purchasing the instrument."

Multiplexing Fluorescence and Luminescence Assays

The ability to simultaneously analyze more that one parameter in a single sample is highly desirable. It allows the researcher to save money and time, while conserving sample material. Thus, the option to multiplex cell-based assays enables the researcher to be more productive and efficient in the laboratory (1)(2)
. The GloMax®-Multi+ Detection System with Instinct Software is an ideal platform for measuring fluorescence and luminescence signals, offering peak sensitivity and a broad dynamic range.

To demonstrate the fluorescent and luminescent multiplexing capabilities of the GloMax®-Multi+ Detection System, the ApoTox-Glo™ Triplex Assay was used. The ApoTox-Glo™ Triplex Assay combines three assay chemistries to assess viability, cytotoxicity and apoptosis events in one well. Viability and cytotoxicity are determined simultaneously with the addition of a nonlytic reagent containing fluorogenic substrates, followed by the addition of a lytic reagent containing a luminogenic substrate to determine caspase activity.

Jurkat cells were treated in a 96-well plate with increasing amounts of staurosporine and ionomycin. Staurosporine is a well-known inducer of apoptosis through caspase activation, while ionomycin treatment has been shown to lead to necrosis with no caspase activity. Following a 6-hour treatment at 37°C, 5% CO2, cell health was evaluated using the ApoTox-Glo™ Assay. Fluorescent viability and cytotoxicity using the live-cell marker AFC and dead cell marker Rhodamine 110, respectively, followed by luminescent caspase measurements were taken with the GloMax®-Multi+ Detection System. As doses increased (in 5% CO2), both compounds induced cell death, but only staurosporine induced death through an apoptotic pathway (Figure 1).

Figure 1. Effect of staurosporine (A) and ionomycin (B) on Jurkat cell health was measured using the ApoTox-Glo™ and the GloMax®-Multi+ Detection System with Instinct Software.

Jurkat cells were treated in a 96-well plate with the indicated amount of each compound. Following a 6-hour treatment at 37°C, 5% CO2, cell health was evaluated using ApoTox-Glo™ Assay. Fluorescent (Rhodamine 110 dead cell marker and AFC live cell marker) and luminescent caspase measurements were taken with the GloMax®-Multi+ Detection System. Each data point is the average of 4 replicates.

Absorbance Measurements at UV Wavelengths

To demonstrate the sensitivity of the absorbance module at UV wavelengths, the lowest detection limit for DNA using the GloMax®-Multi+ absorbance protocol was determined. Genomic DNA stock was serially diluted 1:2 in Nuclease-Free Water to obtain a range of DNA concentrations between 100ng/µl and 0.1ng/µl. One hundred microliters of each DNA concentration was then added to a UV-compatible 96-well plate in quadruplicate. Forty-eight DNA samples were analyzed on the GloMax®-Multi+ using the absorbance protocol, measuring at A260nm and A280nm, in less than 2½ minutes. The results are shown in Figure 2. The detection limit for DNA absorbance was found to be ~1ng/µl, as the data began to show a loss of linearity below 1.55ng/µl (Figure 2). For the entire range of DNA concentrations from 100ng/µl down to 0.1ng/µl, A260 and A280 readings were linear, with R2 values >0.999. This shows that the GloMax®-Multi+ Instrument offers a highly sensitive and easy-to-use absorbance protocol, enabling fast processing of multiple samples at different wavelengths.

Figure 2. Detection of genomic DNA at 260nm and 280nm using the GloMax®-Multi+ Detection System with Instinct Software.

Genomic DNA was serially diluted in quadruplicate in a UV 96-well plate (Costar Cat.# 3635). Sample volumes were 100µl each. Absorbance was measured at 260nm and 280nm using the GloMax®-Multi+ Detection System. A260 and A280 readings are the average ± SEM.

Use of Heating and Shaking Features to Perform a BCA Protein Assay

To demonstrate the shaking, heating and colorimetric detection capabilities of the GloMax®-Multi+ Detection System, a BCA protein assay was performed. The Pierce® BCA Protein Assay Kit was used to detect and quantitate total protein. An albumin (BSA) standard was prepared by diluting the stock BSA from 2,000µg/ml to 3.125µg/ml. Twenty-five microliters of each dilution was added to a 96-well microplate in quadruplicate. Two-hundred microliters of the BCA Working Reagent was added to each sample and placed in the GloMax®-Multi+ Detection System. The samples were shaken in orbital mode for 30 seconds and then incubated at 37°C for 30 minutes within the GloMax®-Multi+ Detection Instrument. Following the incubation, absorbance was measured using the GloMax® UV-Visible absorbance module at 560nm. The results of the BCA Protein Assay using the diluted BSA standard are shown in Table 1 and Figure 3. The standard Microplate Procedure for the Pierce® BCA Protein Assay Kit allows sensitivity in the working range of 20–2,000µg/ml. Using the GloMax®-Multi+ Detection System, we were able to obtain sensitivity as low as 12.5µg/ml. The r2 value for the entire range of the BSA standard was 0.9888. Removing the data point at the uppermost end of the suggested range at 2,000µg/ml improves the r2 value to 0.9985 (data not shown). Thus, the BCA Protein Assay provides a demonstration of the shaking, heating and UV-visible detection capabilities of the GloMax®-Multi+ Detection System.

Table 1. BSA Standard. The A560 reading is the average of quadruplicate samples. The Standard Error of Mean (SEM) is shown.

BSA (µl/ml)

2,000

1,500

1,000

750

500

250

125

25

12.5

6.25

3.125

A560nm

1.879

1.641

1.095

0.842

0.582

0.329

0.170

0.028

0.005

0.000

0.001

SEM

0.069

0.030

0.067

0.026

0.021

0.007

0.005

0.001

0.001

0.002

0.004

Figure 3. Detection of protein using the Pierce® BCA Protein Assay and the GloMax®-Multi+ Detection System with Instinct Software.

A BSA protein standard was prepared from 3.125 to 2,000µg/ml and assayed according to the Pierce® BCA Protein Assay Kit. Twenty-five microliters of each dilution was added to a 96-well clear-bottom plate in quadruplicate. Two hundred microliters of the BCA Working Reagent was added to each sample and placed in the GloMax®-Multi+ Detection System. Samples were shaken in orbital mode for 30 seconds and then incubated at 37°C for 30 minutes within the instrument. Following the incubation, absorbance was measured at 560nm. A560 readings are the average ± SEM.

Summary

The GloMax®-Multi+ Detection System allows researchers the convenience of including all detection requirements in a single instrument. The user-friendly interface enables the easy creation of simple to more complex user-defined protocols. Whether using the luminescence, fluorescence, visible absorbance or UV-visible absorbance or a combination of detection modes, the versatility of the GloMax®-Multi+ Detection System with Instinct Software saves time, money and space by offering these options in one instrument with a customizable format that does not sacrifice the sensitivity and dynamic range available with single-mode instruments.

GloMax is a registered trademark of Promega Corporation. Pierce is a registered trademark of ThermoFisher Scientific.

Figures

Figure 1. Effect of staurosporine (A) and ionomycin (B) on Jurkat cell health was measured using the ApoTox-Glo™ and the GloMax®-Multi+ Detection System with Instinct Software.

Jurkat cells were treated in a 96-well plate with the indicated amount of each compound. Following a 6-hour treatment at 37°C, 5% CO2, cell health was evaluated using ApoTox-Glo™ Assay. Fluorescent (Rhodamine 110 dead cell marker and AFC live cell marker) and luminescent caspase measurements were taken with the GloMax®-Multi+ Detection System. Each data point is the average of 4 replicates.

Figure 2. Detection of genomic DNA at 260nm and 280nm using the GloMax®-Multi+ Detection System with Instinct Software.

Genomic DNA was serially diluted in quadruplicate in a UV 96-well plate (Costar Cat.# 3635). Sample volumes were 100µl each. Absorbance was measured at 260nm and 280nm using the GloMax®-Multi+ Detection System. A260 and A280 readings are the average ± SEM.

Figure 3. Detection of protein using the Pierce® BCA Protein Assay and the GloMax®-Multi+ Detection System with Instinct Software.

A BSA protein standard was prepared from 3.125 to 2,000µg/ml and assayed according to the Pierce® BCA Protein Assay Kit. Twenty-five microliters of each dilution was added to a 96-well clear-bottom plate in quadruplicate. Two hundred microliters of the BCA Working Reagent was added to each sample and placed in the GloMax®-Multi+ Detection System. Samples were shaken in orbital mode for 30 seconds and then incubated at 37°C for 30 minutes within the instrument. Following the incubation, absorbance was measured at 560nm. A560 readings are the average ± SEM.

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